Primer degenerate

Underlined sequences indicate amino acid sequences used for the generation of degenerate primers. Bracketed question marks represent blank cycles from the Edman degradation reaction. Additional sequence was obtained after blank cycles in all cases except the Glu-C-1 and Glu-C-2 peptides. 

The translation of the sequence of the cDNA encoding deacetylvindoline 4-O-acetyltransferase compared to other putative plant acetyltransferases revealed a conserved region near the carboxy terminus of the proteins. This sequence was used to design a degenerate antisense oligodeoxynucleotide primer for PCR. The sense primer was based upon an internal peptide sequence of salutaridinol 7-0-acetyltransferase. This approach finally yielded a partial cDNA that encoded salutaridinol 7-O-acetyltransferase. The full-length clone was obtained by RACE-PCR and was functionally expressed in S. frugiperda Sf9 cells.28 The amino acid sequence of salutaridinol 7-O-acetyltransferase is most similar (37% identity) to that of deacetylvindoline acetyltransferase of C. roseus.27... 

Figure 4. Alignment of the transmembrane domains 3

Fietto, J.L., DeMarco, R. and Verjovski-Almeida, S. (2002) Use of degenerate primers and touchdown PCR for construction of cDNA libraries. Biotechniques 32, 1404-1411. 

Aloe (Aloe arborescens) is a medicinal plant rich in aromatic polyketides such as pharmaceutically important aloenin (a hexaketide pyrone), aloesin (a heptaketide chromone), and barbaloin (an octaketide anthrone) (Fig. 4a). Pentaketide chromone synthase (PCS) and octaketide synthase (OKS) are novel plant-specific type III PKSs, which were obtained from the aloe plant by RT-PCR cloning using degenerate oligonucleotide primers based on the conserved sequences of known CHS enzymes [30-33]. The deduced amino acid sequences of PCS and OKS are 91% identical (368/403), and show 50-60% identity to those of other CHS superfamily type III PKSs of plant origin OKS shares 60% identity (240/403) with CHS from... 

Fig. 3. Universal primers for PCR amplification of endogenous retroviral reverse transcriptase. The primers anneal within the retroviral reverse transcriptase gene at the position indicated by the thick lines in Fig. 2. The degenerate primer pair has been used successfully,21 but it gives a number of nonspecific products. Substitution of inosine at some of the degenerate positions increases the effective concentration of primer. Xba and coRI restriction endonuclease cleavage sites are underlined.

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